.sup.32 P-labeled nucleotides are used routinely for radioactive labeling of DNA and RNA, usually for nucleic acid sequencing and recombinant DNA/RNA research.
Until about 1979, the method generally used for synthesis of radiolabeled nucleotides was via the process disclosed by Schendel and Wells (J. Biol. Chem. 248, 8319-8321 (1973)) which introduced .sup.32 P into one of many well known metabolic pathways as follows: ##STR1##
This pathway, however, does not produce carrier-free nucleotides because the starting material glyceraldehyde-3-phosphate (GAP) is somewhat unstable and hence breaks down to liberate free nonradioactive phosphate, resulting in an apparent specific activity of 4000-6000 Ci/m mole, about half of the theoretical carrier-free specific activity of 9140 Ci/m mole.
To overcome the stability problems associated with utilizing GAP as a starting material, Johnson and Walseth disclosed in U.S. Pat. No. 4,209,589 the concept of generating GAP in situ by tracing backwards in the same metabolic pathway to the starting material glycerophosphate, such process represented by the following pathway: ##STR2##
The Johnson and Walseth method, however, generally only produces [.gamma.-.sup.32 P-labeled] nucleotides having specific activities of from about 4500-6500 Ci/m mole because the enzymes used in the process are only available containing varying amounts of impurities; and purification techniques for these enzymes are quite tedious.
It would be useful, therefore, to devise a simple enzymatic method, preferably one not requiring tedious purification steps, for producing radiolabeled nucleotides that have specific activities approaching theoretical carrier-free levels in order to increase the apparent sensitivity of assays which employ such radiolabeled nucleotides.